Genetically modified methylobacillus bacteria having improving properties

ABSTRACT

The present invention generally relates to the biotechnology engineering, and specifically to genetically modified bacteria of the genus  Methylobacillus  which have improved properties making them particularly useful in large scale methanol fermentations. More specifically, the present invention provides a bacterium of the genus  Methylobacillus  which has been modified to have a decreased production of exopolysaccharides (EPS) compared to an otherwise identical bacterium that does not carry said modification. The present invention further provides a method for producing a biochemical compound using a genetically modified bacterium of the present invention.

TECHNICAL FIELD OF THE INVENTION

The present invention generally relates to the biotechnology engineering, and specifically to genetically modified bacteria of the genus Methylobacillus which have improved properties making them particularly useful in large scale methanol fermentations. More specifically, the present invention provides a bacterium of the genus Methylobacillus which has been modified to have a decreased production of exopolysaccharides (EPS) compared to an otherwise identical bacterium that does not carry said modification. The present invention further provides a method for producing a biochemical compound using a genetically modified bacterium of the present invention.

BACKGROUND OF THE INVENTION

Methanol fermentation has the potential to replace oil-derived chemical production. To realize this, efficient methylotrophic (consuming one-carbon compounds such as methanol or methane or multi-carbon compounds that contain no carbon-carbon bonds) strains must be developed. Several bacteria capable of utilizing methanol as the sole source of carbon for growth and product formation have been identified. Some examples include Bacillus methanolicus, Methylobacterium extorquens, Methylobacillus glycogenes and Methylobacillus flagellatus. Methylotrophic bacteria can be divided into two broad groups based on their methanol assimilation pathways. The first step is always the conversion of methanol into formaldehyde, catalyzed by a methanol dehydrogenase enzyme. In the first group, formaldehyde reacts with Ribulose-monophosphate (RuMP) to form C6-compounds that are then metabolized further. Members of this group are commonly referred to as “RuMP cycle methylotrophs”. The second group, which includes M. extorquens, assimilate formaldehyde by reacting it with glycine, forming serine in the pathway known as the serine cycle. Both groups of methylotrophic bacteria have been used for the production of various biochemicals from methanol. However, the RuMP cycle is more energy efficient than the serine cycle, resulting in faster growth and biomass formation.

Methylotrophic microorganisms can be found in extremely diverse environments, such as oceans, soil, plant rhizosphere and even sewage water. Since they must be capable of surviving these diverse and often harsh environments, methylotrophic bacteria often tend to be quite robust and resistant to different types of stress, all while growing efficiently on minimal nutrients. Strains from the RuMP cycle-utilizing Methylobacillus genus possess a number of traits desired in biochemical productions trains; they grow rapidly and reproducibly in minimal mineral media and they are insensitive to fluctuations in temperature and methanol concentration. Furthermore, they are very amenable to genetic manipulation, unlocking the potential for metabolic engineering of complex pathways.

Many methylotrophs, including Methylobacilli produce large amounts of exopolysaccharides (EPS), which confer multiple advantages to the organisms in their natural environment, such as protection from dessication, biofilm scaffold formation, energy reserves etc. In fed-batch fermentations, Mehtylobacillus biomass can consist of up to 30% EPS.

EPS production in Methylobacilli specifically is relatively unstudied. Research has only been performed on a single strain, Methylobacillus sp. strain 12S, an obligate methylotroph isolated from soil, which was found to synthesize a novel EPS termed methanolan (Yoshida et al., 2000). While the gene cluster responsible for methanolan synthesis in Methylobacillus sp. strain 12S has been identified and characterized (Yoshida et al., 2003), no such work has been done in other Methylobacilli. Based on bioinformatic analysis and sequence similarities, an EPS gene cluster has been predicted in Methylobacillus flagellatus strain KT (Chistoserdova et al., 2007) and Methylobacillus glycogenes (Hattori et al., 2020), though the genes themselves have never been characterized.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome certain drawbacks in methanol-based bioprocesses. This is achieved by the present inventors who have engineered different genetically modified bacteria of the genus Methylobacillus having a decreased production of exopolysaccharides (EPS).

The present inventors studied general EPS production in Methylobacilli. Based on bioinformatic analysis of the genome of M. flagellatus and M. glycogenes, the present inventors identified two putative EPS-producing gene clusters. Disruption of both EPS clusters resulted in complete abolishment of EPS production.

An unexpected and surprising observation in the modified M. flagellatus and M. glycogenes strains was their behavior in growth cultures. When abolishing the expression of one or more endogenous polypeptides involved in the production of exopolysaccharides (EPS) in the Methylobacillus bacteria, the culture showed a number of surprising and unexpected properties beneficial to the development of methanol-based bioprocesses. Unexpectedly, the excessive culture foaming in both shake flasks and bioreactors was reduced. Moreover, while cell clumping was observed for unmodified parent strains, this was reduced or even absent in the culture of modified Methylobacilli. Centrifugation and filtration of the biomass was also improved when EPS production was eliminated. These combined characteristics significantly improve the bioprocess, strain handling, and downstream processing, making modified Methylobacillus bacteria suitable for large-scale methanol fermentations. Removal of EPS synthesis unexpectedly also improved the methanol tolerance of M. flagellatus and other Methylobacillus species.

Based on the foregoing finds, the present invention provides in a first aspect a genetically engineered bacterium of the genus Methylobacillus which has been modified to have a decreased production of exopolysaccharides (EPS) compared to an otherwise identical bacterium that does not carry said modification.

The present invention further provides a method for the production of a biochemical compound comprising cultivating a bacterium of the present invention under suitable culture conditions in a culture medium comprising a reduced one-carbon compound, such as methanol, or a multi-carbon compound that contains no carbon-carbon bonds, such as dimethylamine.

The present invention may be summarized by the following items:

-   -   1. A genetically engineered bacterium of the genus         Methylobacillus which has been modified to have a decreased         production of exopolysaccharides (EPS) compared to an otherwise         identical bacterium that does not carry said modification.     -   2. The bacterium according to item 1, which has been modified to         have a decreased expression and/or function (e.g. activity) of         at least one endogenous polypeptide involved in the production         of exopolysaccharides (EPS) in said bacterium compared to an         otherwise identical bacterium that does not carry said         modification (reference bacterium).     -   3. The bacterium according to item 1 or 2, which has been         modified to have a decreased expression of at least one         endogenous polypeptide involved in the production of         exopolysaccharides (EPS) in said bacterium compared to an         otherwise identical bacterium that does not carry said         modification (reference bacterium).     -   4. The bacterium according to any one of items 1 to 3, which has         been modified to have a decrease expression of at least two         endogenous polypeptides involved in the production of         exopolysaccharides (EPS) in said bacterium compared to an         otherwise identical bacterium that does not carry said         modification (reference bacterium).     -   5. The bacterium according to any one of items 1 to 4, which has         been modified to have a decrease expression of at least three         endogenous polypeptides involved in the production of         exopolysaccharides (EPS) in said bacterium compared to an         otherwise identical bacterium that does not carry said         modification (reference bacterium).     -   6. The bacterium according to any one of items 1 to 5, which has         been modified to have a decrease expression of at least four         endogenous polypeptides involved in the production of         exopolysaccharides (EPS) in said bacterium compared to an         otherwise identical bacterium that does not carry said         modification (reference bacterium).     -   7. The bacterium according to any one of items 1 to 6, which has         been modified to have a decrease expression of at least five         endogenous polypeptides involved in the production of         exopolysaccharides (EPS) in said bacterium compared to an         otherwise identical bacterium that does not carry said         modification (reference bacterium).     -   8. The bacterium according to any one of items 1 to 7, which has         been modified to have a decrease expression of at least six         endogenous polypeptides involved in the production of         exopolysaccharides (EPS) in said bacterium compared to an         otherwise identical bacterium that does not carry said         modification (reference bacterium).     -   9. The bacterium according to any one of items 1 to 8, which has         been modified to have a decrease expression of all endogenous         polypeptides involved in the production of exopolysaccharides         (EPS) in said bacterium compared to an otherwise identical         bacterium that does not carry said modification (reference         bacterium).     -   10. The bacterium according to any one of items 2 to 9, wherein         the at least one polypeptide involved in the production of         exopolysaccharides (EPS) in said bacterium is selected from the         group consisting of a) polypeptides comprising an amino acid         sequence set forth in any one of SEQ ID NO: 1 to 38 and b)         polypeptides comprising an amino acid sequence having at least         70% sequence identity with any one of SEQ ID NO: 1 to 38 and         having the same functional property as the reference         polypeptide.     -   11. The bacterium according to any one of items 2 to 10, wherein         the at least one polypeptide involved in the production of         exopolysaccharides (EPS) in said bacterium is selected from the         group consisting of a) polypeptides comprising an amino acid         sequence set forth in any one of SEQ ID NO: 1 to 26 and b)         polypeptides comprising an amino acid sequence having at least         70% sequence identity with any one of SEQ ID NO: 1 to 26 and         having the same functional property as the reference         polypeptide.     -   12. The bacterium according to any one of items 2 to 10, wherein         the at least one polypeptide involved in the production of         exopolysaccharides (EPS) in said bacterium is selected from the         group consisting of a) polypeptides comprising an amino acid         sequence set forth in any one of SEQ ID NO: 27 to 38 and b)         polypeptides comprising an amino acid sequence having at least         70% sequence identity with any one of SEQ ID NO: 27 to 38 and         having the same functional property as the reference         polypeptide.     -   13. The bacterium according to any one of items 2 to 12, wherein         the at least one polypeptide involved in the production of         exopolysaccharides (EPS) in said bacterium is selected from the         group consisting of a) polypeptides comprising an amino acid         sequence set forth in any one of SEQ ID NO: 39 to 86 and b)         polypeptides comprising an amino acid sequence having at least         70% sequence identity with any one of SEQ ID NO: 39 to 86 and         having the same functional property as the reference         polypeptide.     -   14. The bacterium according to any one of items 2 to 13, wherein         the at least one polypeptide involved in the production of         exopolysaccharides (EPS) in said bacterium is selected from the         group consisting of a) polypeptides comprising an amino acid         sequence set forth in any one of SEQ ID NO: 39 to 60 and b)         polypeptides comprising an amino acid sequence having at least         70% sequence identity with any one of SEQ ID NO: 39 to 60 and         having the same functional property as the reference         polypeptide.     -   15. The bacterium according to any one of items 2 to 13, wherein         the at least one polypeptide involved in the production of         exopolysaccharides (EPS) in said bacterium is selected from the         group consisting of a) polypeptides comprising an amino acid         sequence set forth in any one of SEQ ID NO: 61 to 86 and b)         polypeptides comprising an amino acid sequence having at least         70% sequence identity with any one of SEQ ID NO: 61 to 86 and         having the same functional property as the reference         polypeptide.     -   16. The bacterium according to any one of items 2 to 15, wherein         the at least one polypeptide involved in the production of         exopolysaccharides (EPS) in said bacterium is selected from the         group consisting a polypeptide having peptidyl-prolyl cis-trans         isomerase activity, a polypeptide capable of acting as a         polysaccharide exporter; a polypeptide acting as a chain length         determinant protein; and a polypeptide having protein-tyrosine         kinase activity.     -   17. The bacterium according to item 16, wherein the polypeptide         having peptidyl-prolyl cis-trans isomerase activity comprises an         amino acid sequence having at least 70% sequence identity to SEQ         ID NO: 6, the polypeptide capable of acting as a polysaccharide         exporter comprises an amino acid sequence having at least 70%         sequence identity to SEQ ID NO: 7, the polypeptide acting as a         chain length determinant protein comprises an amino acid         sequence having at least 70% sequence identity to SEQ ID NO: 8         and the polypeptide having protein-tyrosine kinase activity         comprises an amino acid sequence having at least 70% sequence         identity to SEQ ID NO: 9.     -   18. The bacterium according to item 16, wherein the polypeptide         having peptidyl-prolyl cis-trans isomerase activity comprises an         amino acid sequence having at least 70% sequence identity to SEQ         ID NO: 42, the polypeptide capable of acting as a polysaccharide         exporter comprises an amino acid sequence having at least 70%         sequence identity to SEQ ID NO: 43, the polypeptide acting as a         chain length determinant protein comprises an amino acid         sequence having at least 70% sequence identity to SEQ ID NO: 44         and the polypeptide having protein-tyrosine kinase activity         comprises an amino acid sequence having at least 70% sequence         identity to SEQ ID NO: 45.     -   19. The bacterium according to any one of items 2 to 18, wherein         the expression of the at least one polypeptide involved in the         production of exopolysaccharides (EPS) in said bacterium is         decreased by at least 50%, such as by at least 60%, at least         65%, at least 70%, at least 75%, at least 80%, at least 85%, at         least 90%, at least 95% or at least 100% compared to the         otherwise identical bacterium.     -   20. The bacterium according to any one of items 2 to 19, wherein         the expression of the at least one polypeptide involved in the         production of exopolysaccharides (EPS) in said bacterium is         abolished compared to the otherwise identical bacterium.     -   21. The bacterium according to any one of items 2 to 20, wherein         the endogenous gene/s encoding said polypeptide/s involved in         the production of exopolysaccharides (EPS) in said bacterium         has/have been inactivated.     -   22. The bacterium according to any one of items 2 to 21, wherein         the endogenous gene/s encoding said polypeptides/s involved in         the production of exopolysaccharides (EPS) in said bacterium         has/have been inactivated by deletion of part of or the entire         gene sequence.     -   23. The bacterium according to any one of items 2 to 22, wherein         the endogenous gene/s encoding said polypeptide/s involved in         the production of exopolysaccharides (EPS) in said bacterium         has/have been inactivated by introducing or expressing in the         bacterium a respective rare-cutting endonuclease able to         selectively inactivating by DNA cleavage the endogenous gene         encoding said polypeptide.     -   24. The bacterium according to item 23, wherein said         rare-cutting endonucleases are selected from the group         consisting of a transcription activator-like effector (TALE)         nuclease, meganuclease, zing-finger nuclease (ZFN) and         RNA-guided endonuclease.     -   25. The bacterium according to item 24, wherein the RNA-guided         endonuclease is a catalytically inactive Cas9 protein.     -   26. The bacterium according to item 25, which comprises (e.g.,         expresses) at least one single guide RNA (sgRNA) specifically         hybridizing (e.g. binding) under cellular conditions with the         genomic DNA encoding said enzyme(s).     -   27. The bacterium according to any one of items 2 to 21, wherein         the expression of the polypeptide(s) involved in the production         of exopolysaccharides (EPS) in said bacterium is decreased         (e.g., inhibited) by transcriptional and/or translational         repression of the endogenous gene(s) encoding said         polypeptide(s).     -   28. The bacterium according to any one of items 2 to 21, wherein         the expression of the polypeptide(s) involved in the production         of exopolysaccharides (EPS) in said bacterium is decreased         (e.g., inhibited) by introducing or expressing in the bacterium         at least one inhibitory nucleic acid molecule that specifically         hybridizes (e.g. binds) under cellular conditions with cellular         mRNA and/or genomic DNA encoding said polypeptide(s).     -   29. The bacterium according to item 28, wherein the inhibitory         nucleic acid molecule is an antisense oligonucleotide, ribozyme         or interfering RNA (RNAi) molecule.     -   30. The bacterium according to item 29, wherein the interfering         RNA molecule is a micro RNA (miRNA), small interfering RNA         (siRNA) or short hairpin RNA (shRNA).     -   31. The bacterium according to any one of items 2 to 30, wherein         the endogenous polypeptide or polypeptides involved in the         production of exopolysaccharides (EPS) in said bacterium is/are         encoded by a gene or genes included in a first EPS gene cluster         and/or second EPS gene cluster.     -   32. The bacterium according to item 31, wherein the first EPS         gene cluster includes genes defined by or orthologous to the         open reading frames (ORFS) found in SEQ ID NO: 174 or 177.     -   33. The bacterium according to item 31 or 32, wherein the first         EPS gene cluster includes the genes epsD, epsE, epsF, epsG,         epsB, epsL, epsH, epsI, epsJ and epsS, or orthologs thereof.     -   34. The bacterium according to any one of items 31 to 33,         wherein the first EPS gene cluster includes the genes epsD,         epsE, epsF and epsG, or orthologs thereof.     -   35. The bacterium according to any one of items 31 to 34,         wherein the first EPS gene cluster includes a nucleotide         sequence which has at least 50% sequence identity with the         nucleotide sequence of SEQ ID NO: 174 or 177.     -   36. The bacterium according to any one of items 31 to 34,         wherein the first EPS gene cluster includes a nucleotide         sequence which has at least 50% sequence identity with the         nucleotide sequence of SEQ ID NO: 174.     -   37. The bacterium according to any one of items 31 to 36,         wherein at least one gene in said first EPS gene cluster is         inactivated, e.g., by deletion of part of or the entire gene         sequence.     -   38. The bacterium according to any one of items 31 to 37,         wherein at least one gene selected from epsD, epsE, epsF, epsG,         epsB, epsL, epsH, epsI, epsJ and epsS, or ortholog thereof, is         inactivated, e.g., by deletion of part of or the entire gene         sequence.     -   39. The bacterium according to any one of items 31 to 38,         wherein at least one gene selected from epsD, epsE, epsF and         epsG, or ortholog thereof, is inactivated, e.g., by deletion of         part of or the entire gene sequence.     -   40. The bacterium according to any one of items 31 to 39,         wherein the genes epsD, epsE, epsF and epsG, or orthologs         thereof, are inactivated, e.g., by deletion of part of or the         entire gene sequence.     -   41. The bacterium according to item 39 or 40, wherein the gene         epsD encodes a polypeptide comprising an amino acid sequence         having at least 70% sequence identity to SEQ ID NO: 6, wherein         the gene epsE encodes a polypeptide comprising an amino acid         sequence having at least 70% sequence identity to SEQ ID NO: 7,         wherein the gene epsF encodes a polypeptide comprising an amino         acid sequence having at least 70% sequence identity to SEQ ID         NO: 8, and wherein the gene epsG encodes a polypeptide         comprising an amino acid sequence having at least 70% sequence         identity to SEQ ID NO: 9.     -   42. The bacterium according to item 41, wherein the gene epsD         comprises a nucleotide sequence having at least 70% sequence         identity with SEQ ID NO: 92, the gene epsE comprises a         nucleotide sequence having at least 70% sequence identity with         SEQ ID NO: 93, the gene epsF comprises an nucleotide sequence         having at least 70% sequence identity with SEQ ID NO: 94, and         the gene epsG comprises an nucleotide sequence having at least         70% sequence identity with SEQ ID NO: 95.     -   43. The bacterium according to item 39 or 40, wherein the gene         epsD encodes a polypeptide comprising an amino acid sequence         having at least 70% sequence identity to SEQ ID NO: 42, wherein         the gene epsE encodes a polypeptide comprising an amino acid         sequence having at least 70% sequence identity to SEQ ID NO: 43,         wherein the gene epsF encodes a polypeptide comprising an amino         acid sequence having at least 70% sequence identity to SEQ ID         NO: 44, and wherein the gene epsG encodes a polypeptide         comprising an amino acid sequence having at least 70% sequence         identity to SEQ ID NO: 45.     -   44. The bacterium according to item 43, wherein the epsD gene         comprises a nucleotide sequence having at least 70% sequence         identity with SEQ ID NO: 128, the gene epsE comprises a         nucleotide sequence having at least 70% sequence identity with         SEQ ID NO: 129, the gene epsF comprises an nucleotide sequence         having at least 70% sequence identity with SEQ ID NO: 130, and         the gene epsG comprises an nucleotide sequence having at least         70% sequence identity with SEQ ID NO: 131.     -   45. The bacterium according to any one of items 31 to 44,         wherein the first EPS gene cluster is inactivated.     -   46. The bacterium according to any one of items 31 to 45,         wherein the first EPS gene cluster is inactivated by deletion of         part of or the entire sequence of said cluster.     -   47. The bacterium according to any one of items 31 to 45,         wherein the first EPS gene cluster is inactivated by         modification of (e.g., by introducing at least one mutation in)         the promoter and/or ribosome binding site region resulting in         the lack of gene expression.     -   48. The bacterium according to any one of items 31 to 47,         wherein the second EPS gene cluster includes genes defined by or         orthologous to the open reading frames (ORFS) found in SEQ ID         NO: 175 or 179.     -   49. The bacterium according to any one of items 31 to 48,         wherein the second EPS gene cluster includes a nucleotide         sequence which has at least 50% sequence identity with the         nucleotide sequence of 175 or 179.     -   50. The bacterium according to any one of items 31 to 48,         wherein the second EPS gene cluster includes a nucleotide         sequence which has at least 50% sequence identity with the         nucleotide sequence of 175.     -   51. The bacterium according to any one of items 31 to 50,         wherein at least one gene in said second EPS gene cluster is         inactivated, e.g., by deletion of part of or the entire gene         sequence.     -   52. The bacterium according to any one of items 31 to 51,         wherein the second EPS gene cluster is inactivated.     -   53. The bacterium according to any one of items 31 to 52,         wherein the second EPS gene cluster is inactivated by deletion         of part of or the entire sequence of said cluster.     -   54. The bacterium according to any one of items 31 to 52,         wherein the second EPS gene cluster is inactivated by         modification of (e.g., by introducing at least one mutation in)         the promoter and/or ribosome binding site region resulting in         the lack of gene expression.     -   55. The bacterium according to any one of 1 to 52, wherein all         endogenous genes encoding enzymes involved in the production of         exopolysaccharides (EPS) in said bacterium are inactivated,         e.g., deleted.     -   56. The bacterium according to any one of items 1 to 55, wherein         the bacterium is selected from Methylobacillus flagellatus,         Methylobacillus glycogenes, Methylobacillus pratensis,         Methylobacillus rhizosphaerae, Methylobacillus gramineus,         Methylobacillus arboreus, Methylobacillus caricics,         Methylobacillus methilovorans and Methylobacillus sp.     -   57. The bacterium according to any one of items 1 to 56, wherein         the bacterium is selected from Methylobacillus flagellatus and         Methylobacillus glycogenes.     -   58. The bacterium according to any one of items 1 to 55, wherein         the bacterium is Methylobacillus flagellatus.     -   59. The bacterium according to any one of items 1 to 55, wherein         the bacterium is Methylobacillus glycogenes.     -   60. A method for the production of a biochemical compound         comprising cultivating a bacterium according to any one of items         1 to 59 under suitable culture conditions in a culture medium         comprising a reduced one-carbon compound, such as methanol, or a         multi-carbon compound that contains no carbon-carbon bonds, such         as dimethylamine.     -   61. The method according to item 60, wherein the culture medium         comprises methanol.     -   62. The method according to item 60 or 61, wherein the         cultivation is performed in a bioreactor.     -   63. The method according to any one of items 60 to 62, wherein         the biochemical compound is selected from organic acids, amino         acids, fatty acids and derivatives thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 : Sugar content of hydrolyzed M. flagellatus shake-flask supernatant. Dark gray bars represent the total glucose in mg/L and the light gray bars represent total monomeric sugars. Error bars are the standard deviation of technical replicates.

FIG. 2 : Measured viscosity of bioreactor broth produced by different strains of Methylobacillus flagellatus.

FIG. 3 : Photo of wild type M. flagellatus and M. glycogenes cultures after 24 hours of incubation. Wild type ABME 5 and 6 on the left, followed by a single-cluster disruption in M. flagellatus and finally a double disruption.

FIG. 4 : Plot of antifoam addition (y axis) during two representative bioreactor fermentations of ABME 6 and ABME 131 in relation to fermentation time (x axis).

FIG. 5 : Phase-contrast microscopy images of bioreactor fermentation broth of wild type Methylobacillus flagellatus (left) vs the double-disruption strain ABME 131 (right).

FIG. 6 : Improved growth of the EPS free ABM E131 compared to the parent strain.

The present invention is now described in more detail below.

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of biochemistry, genetics, and microbiology.

All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, and recombinant DNA, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “Gene Expression Technology” (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory).

Bacterium of the Invention

As indicated above, the present invention is based on the unexpected and surprising finding that certain drawbacks in methanol-based bioprocesses can be overcome by down-regulating the production of exopolysaccharides (EPS) in bacteria of the genus Methylobacillus.

The present invention thus provides in a first aspect a genetically engineered bacterium of the genus Methylobacillus which has been modified to have a decreased production of exopolysaccharides (EPS) compared to an otherwise identical bacterium that does not carry said modification.

More specifically, the present invention provides a genetically engineered bacterium of the genus Methylobacillus which has been modified to have a decreased expression and/or activity of at least one endogenous polypeptide involved in the production of exopolysaccharides (EPS) in said bacterium compared to an otherwise identical bacterium that does not carry said modification (reference bacterium).

According to some embodiments, the genetically engineered bacterium of the invention has been modified to have a decreased expression of at least one, such as at least two, endogenous polypeptide involved in the production of exopolysaccharides (EPS) in said bacterium compared to an otherwise identical bacterium that does not carry said modification (reference bacterium).

According to some embodiments, the genetically engineered bacterium of the invention has been modified to have a decreased expression of at least three, such as at least four, endogenous polypeptides involved in the production of exopolysaccharides (EPS) in said bacterium compared to an otherwise identical bacterium that does not carry said modification (reference bacterium).

According to some embodiments, the genetically engineered bacterium of the invention has been modified to have a decreased expression of at least five, such as at least six, endogenous polypeptides involved in the production of exopolysaccharides (EPS) in said bacterium compared to an otherwise identical bacterium that does not carry said modification (reference bacterium).

According to some embodiments, the genetically engineered bacterium of the invention has been modified to have a decreased expression of at least seven, such as at least eight, endogenous polypeptides involved in the production of exopolysaccharides (EPS) in said bacterium compared to an otherwise identical bacterium that does not carry said modification (reference bacterium).

According to some embodiments, the genetically engineered bacterium of the invention has been modified to have a decreased expression of at least nine, such as at least ten, endogenous polypeptides involved in the production of exopolysaccharides (EPS) in said bacterium compared to an otherwise identical bacterium that does not carry said modification (reference bacterium).

According to some embodiments, the genetically engineered bacterium of the invention has been modified to have a decreased expression of at least eleven, such as at least twelve, endogenous polypeptides involved in the production of exopolysaccharides (EPS) in said bacterium compared to an otherwise identical bacterium that does not carry said modification (reference bacterium).

According to some embodiments, the genetically engineered bacterium of the invention has been modified to have a decreased expression of at least thirteen, such as at least fourteen, endogenous polypeptides involved in the production of exopolysaccharides (EPS) in said bacterium compared to an otherwise identical bacterium that does not carry said modification (reference bacterium).

According to some embodiments, the genetically engineered bacterium of the invention has been modified to have a decreased expression of at least fifteen, such as at least sixteen, endogenous polypeptides involved in the production of exopolysaccharides (EPS) in said bacterium compared to an otherwise identical bacterium that does not carry said modification (reference bacterium).

According to some embodiments, the genetically engineered bacterium of the invention has been modified to have a decreased expression of all endogenous polypeptides involved in the production of exopolysaccharides (EPS) in said bacterium compared to an otherwise identical bacterium that does not carry said modification (reference bacterium).

According to some embodiments, the at least one polypeptide involved in the production of exopolysaccharides (EPS) in said bacterium is selected from the group consisting of a) polypeptides comprising an amino acid sequence set forth in any one of SEQ ID NO: 1 to 38 and b) polypeptides comprising an amino acid sequence having at least 70%%, such as at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, sequence identity with any one of SEQ ID NO: 1 to 38 and having the same functional property as the reference polypeptide.

According to some embodiments, the at least one polypeptide involved in the production of exopolysaccharides (EPS) in said bacterium is selected from the group consisting of a) polypeptides comprising an amino acid sequence set forth in any one of SEQ ID NO: 1 to 26 and b) polypeptides comprising an amino acid sequence having at least 70%%, such as at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, sequence identity with any one of SEQ ID NO: 1 to 26 and having the same functional property as the reference polypeptide.

According to some embodiments, the at least one polypeptide involved in the production of exopolysaccharides (EPS) in said bacterium is selected from the group consisting of a) polypeptides comprising an amino acid sequence set forth in any one of SEQ ID NO: 27 to 38 and b) polypeptides comprising an amino acid sequence having at least 70%%, such as at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, sequence identity with any one of SEQ ID NO: 27 to 38 and having the same functional property as the reference polypeptide.

According to some embodiments, the at least one polypeptide involved in the production of exopolysaccharides (EPS) in said bacterium is selected from the group consisting of a) polypeptides comprising an amino acid sequence set forth in any one of SEQ ID NO: 39 to 86 and b) polypeptides comprising an amino acid sequence having at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, sequence identity with any one of SEQ ID NO: 39 to 86 and having the same functional property as the reference polypeptide.

According to some embodiments, the the at least one polypeptide involved in the production of exopolysaccharides (EPS) in said bacterium is selected from the group consisting of a) polypeptides comprising an amino acid sequence set forth in any one of SEQ ID NO: 39 to 60 and b) polypeptides comprising an amino acid sequence having at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, sequence identity with any one of SEQ ID NO: 39 to 60 and having the same functional property as the reference polypeptide.

According to some embodiments, the at least one enzyme involved in the production of exopolysaccharides (EPS) in said bacterium is selected from the group consisting of a) polypeptides comprising an amino acid sequence set forth in any one of SEQ ID NO: 61 to 86 and b) polypeptides comprising an amino acid sequence having at least 70%%, such as at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, sequence identity with any one of SEQ ID NO: 61 to 86 and having the same functional property as the reference polypeptide.

According to some embodiments, the at least one polypeptide involved in the production of exopolysaccharides (EPS) in said bacterium is selected from the group consisting a polypeptide having peptidyl-prolyl cis-trans isomerase activity, a polypeptide capable of acting as a polysaccharide exporter; a polypeptide acting as a chain length determinant protein; and a polypeptide having protein-tyrosine kinase activity.

According to some embodiments, the polypeptide having peptidyl-prolyl cis-trans isomerase activity comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6.

According to some embodiments, the polypeptide capable of acting as a polysaccharide exporter comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 7.

According to some embodiments, the polypeptide acting as a chain length determinant protein comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8.

According to some embodiments, the polypeptide having protein-tyrosine kinase activity comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 9.

According to some embodiments, the polypeptide having peptidyl-prolyl cis-trans isomerase activity comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 42.

According to some embodiments, the polypeptide capable of acting as a polysaccharide exporter comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 43.

According to some embodiments, the polypeptide acting as a chain length determinant protein comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 44.

According to some embodiments, the polypeptide having protein-tyrosine kinase activity comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 45.

The expression level of the endogenous polypeptide(s) may, for example, be decreased by at least 50%, such as by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100% compared to the otherwise identical bacterium.

A decrease of the expression of the endogenous polypeptide(s) may be achieved by any suitable means known in the art. For example, the expression may be decrease by inactivating the endogenous gene(s) encoding said polypeptide(s) involved in the production of exopolysaccharides (EPS) in the bacterium, such as by deletion of part of or the entire gene sequence.

According to some embodiments, the expression of the at least one polypeptide involved in the production of exopolysaccharides (EPS) in said bacterium is abolished compared to the otherwise identical bacterium.

According to some embodiments, the endogenous gene/s encoding said polypeptide/s involved in the production of exopolysaccharides (EPS) in the bacterium has/have been inactivated, such as by deletion of part of or the entire gene sequence.

According to some embodiments, the endogenous gene/s encoding said polypeptide/s involved in the production of exopolysaccharides (EPS) in said bacterium has/have been inactivated by introducing or expressing in the bacterium a rare-cutting endonuclease able to selectively inactivating by DNA cleavage the endogenous gene encoding said enzyme. A rare-cutting endonuclease to be used in accordance of the present invention to inactivate the endogenous gene may, for instance, be a transcription activator-like effector (TALE) nuclease, meganuclease, zing-finger nuclease (ZFN), or RNA-guided endonuclease.

One way to inactivate an endogenous gene encoding a polypeptide involved in the production of exopolysaccharides (EPS) in the bacterium is to use the CRISPRi system. The CRISPRi system was developed as a tool for targeted repression of gene expression or for blocking targeted locations on the genome. The CRISPRi system consists of the catalytically inactive, “dead” Cas9 protein (dCas9) and a guide RNA that defines the binding site for the dCas9 to DNA.

According to some embodiments, the expression of an endogenous polypeptide involved in the production of exopolysaccharides (EPS) in the bacterium is decreased by way of inhibition.

Inhibition of the expression of said polypeptide may be achieved by any suitable means known in the art. For example, the expression may be inhibited by gene silencing techniques involving the use of inhibitory nucleic acid molecules, such as antisense oligonucleotides, ribozymes or interfering RNA (RNAi) molecules, such as microRNA (miRNA), small interfering RNA (siRNA) or short hairpin RNA (shRNA).

According to some embodiments, the expression of an endogenous polypeptide involved in the production of exopolysaccharides (EPS) in the bacterium is decreased (e.g., inhibited) by transcriptional and/or translational repression of the endogenous gene encoding said polypeptide.

According to some embodiments, the expression of an endogenous polypeptide involved in the production of exopolysaccharides (EPS) in the bacterium is inhibited by introducing or expressing in the bacterium an inhibitory nucleic acid molecule. For example, the inhibitory nucleic acid molecule may be introduced by way of an exogenous nucleic acid molecule comprising a nucleotide sequence encoding said inhibitory nucleic acid molecule operably linked to a promoter, such as an inducible promoter, that is functional in the bacterium to cause the production of said inhibitory nucleic acid molecule. Suitably, the inhibitory nucleic acid molecule is one that specifically hybridizes (e.g. binds) under cellular conditions with cellular mRNA and/or genomic DNA encoding the endogenous polypeptide in question. Depending on the target, transcription of the encoding genomic DNA and/or translation of the encoding mRNA is/are inhibited.

According to some embodiments, the inhibitory nucleic acid molecule is an antisense oligonucleotide, ribozyme or interfering RNA (RNAi) molecule. Preferably, such nucleic acid molecule comprises at least 10 consecutive nucleotides of the complement of the cellular mRNA and/or genomic DNA encoding the polypeptide in question.

According to some embodiments, the inhibitory nucleic acid is an antisense oligonucleotide. Such antisense oligonucleotide is a nucleic acid molecule (either DNA or RNA) which specifically hybridizes (e.g. binds) under cellular conditions with the cellular mRNA and/or genomic DNA encoding the enzyme in question.

According to some embodiments, the inhibitory nucleic acid molecule is a ribozyme, such as a hammerhead ribozyme. A ribozyme molecule is designed to catalytically cleave the mRNA transcript to prevent translation of the polypeptide in question.

According to some embodiments, the inhibitory nucleic acid molecule is an interfering RNA (RNAi) molecule. RNA interference is a biological process in which RNA molecules inhibit expression, typically causing the destruction of specific mRNA. Exemplary types of RNAi molecules include microRNA (miRNA), small interfering RNA (siRNA) and short hairpin RNA (shRNA). According to some embodiments, the RNAi molecule is a miRNA. According to some embodiments, the RNAi molecule is a siRNA. According to some embodiments, the RNAi molecule is a shRNA.

According to some embodiments, bacterium of the invention has been modified to have a decreased function (e.g. activity) of at least one polypeptide involved in the production of exopolysaccharides (EPS) in the bacterium compared to an otherwise identical microorganism that does not carry said modification (reference bacterium).

A decrease of the function (e.g. activity) of the at least one endogenous polypeptide may be achieved by any suitable means known in the art. For example, if the polypeptide is an enzyme the activity may be decrease by introducing one or more mutations in the active site of the enzyme resulting in the reduction or loss of activity. Thus, according to some embodiments, the activity of the at least one endogenous enzyme involved in the production of exopolysaccharides (EPS) in the bacterium is decreased by at least one active-site mutation resulting in the reduction or loss of activity. The at least one active-site mutation may, for example, be at least one non-conservative amino acid substitution.

As noted above, the present inventors have identified two putative EPS gene clusters in the genome of Methylobacilli. The endogenous polypeptide or polypeptides involved in the production of exopolysaccharides (EPS) is/are thus encoded by a gene or genes included in a first EPS gene cluster and/or second EPS gene cluster.

According to some embodiments, the endogenous polypeptide or polypeptides involved in the production of exopolysaccharides (EPS) in said bacterium is/are encoded by a gene or genes included in a first EPS gene cluster and/or second EPS gene cluster.

According to some embodiments, the first EPS gene cluster includes genes defined by or orthologous to the open reading frames (ORFS) found in SEQ ID NO: 174 or 177.

According to some embodiments, the first EPS gene cluster includes the genes epsD, epsE, epsF, epsG, epsB, epsL, epsH, epsI, epsJ and epsS, or orthologs thereof.

According to some embodiments, the first EPS gene cluster includes the genes epsD, epsE, epsF and epsG, or orthologs thereof.

According to some embodiments, the first EPS gene cluster includes a nucleotide sequence which has at least 50%, such as at least 55%, sequence identity with the nucleotide sequence of SEQ ID NO: 174 or 177.

According to some embodiments, the first EPS gene cluster includes a nucleotide sequence which has at least 60%, such as at least 65%, sequence identity with the nucleotide sequence of SEQ ID NO: 174 or 177.

According to some embodiments, the first EPS gene cluster includes a nucleotide sequence which has at least 70%, such as at least 75%, sequence identity with the nucleotide sequence of SEQ ID NO: 174 or 177.

According to some embodiments, the first EPS gene cluster includes a nucleotide sequence which has at least 80%, such as at least 85%, sequence identity with the nucleotide sequence of SEQ ID NO: 174 or 177.

According to some embodiments, the first EPS gene cluster includes a nucleotide sequence which has at least 90%, such as at least 93%, sequence identity with the nucleotide sequence of SEQ ID NO: 174 or 177.

According to some embodiments, the first EPS gene cluster includes a nucleotide sequence which has at least 95%, such as at least 97%, sequence identity with the nucleotide sequence of SEQ ID NO: 174 or 177.

According to some embodiments, the first EPS gene cluster includes a nucleotide sequence which has at least 50%, such as at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79% at least, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the nucleotide sequence of SEQ ID NO: 174.

According to some embodiments, the first EPS gene cluster includes a nucleotide sequence which has at least 50%, such as at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%at least, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the nucleotide sequence of SEQ ID NO: 177.

According to some embodiments, the second EPS gene cluster includes genes defined by or orthologous to the open reading frames (ORFS) found in SEQ ID NO: 175 or 179.

According to some embodiments, the second EPS gene cluster includes a nucleotide sequence which has at least 50%, such as at least 55%, sequence identity with the nucleotide sequence of 175 or 179.

According to some embodiments, the second EPS gene cluster includes a nucleotide sequence which has at least 60%, such as at least 65%, sequence identity with the nucleotide sequence of 175 or 179.

According to some embodiments, the second EPS gene cluster includes a nucleotide sequence which has at least 70%, such as at least 75%, sequence identity with the nucleotide sequence of 175 or 179.

According to some embodiments, the second EPS gene cluster includes a nucleotide sequence which has at least 80%, such as at least 85%, sequence identity with the nucleotide sequence of 175 or 179.

According to some embodiments, the second EPS gene cluster includes a nucleotide sequence which has at least 90%, such as at least 85%, sequence identity with the nucleotide sequence of 175 or 179.

According to some embodiments, the second EPS gene cluster includes a nucleotide sequence which has at least 50%, such as at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%at least, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the nucleotide sequence of 175.

According to some embodiments, the second EPS gene cluster includes a nucleotide sequence which has at least 50%, such as at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%at least, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with the nucleotide sequence of 179.

According to some embodiments, at least one gene in said first EPS gene cluster is inactivated, e.g., by deletion of part of or the entire gene sequence.

According to some embodiments, the at least one gene is selected from epsD, epsE, epsF, epsG, epsB, epsL, epsH, epsI, epsJ and epsS, or ortholog thereof.

According to some embodiments, the at least one gene is selected from epsD, epsE, epsF and epsG, or ortholog thereof.

According to some embodiments, the genes epsD, epsE, epsF and epsG, or orthologs thereof, are inactivated, e.g., by deletion of part of or the entire gene sequence.

According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 6, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 7, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 8, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 9.

According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 6, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 7, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 80%, such as at least 85, sequence identity to SEQ ID NO: 8, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 9.

According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 6, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 7, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 90%, such as at least 95, sequence identity to SEQ ID NO: 8, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 9.

According to some embodiments, the epsD gene comprises a nucleotide sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 92, the gene epsE comprises a nucleotide sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 93, the gene epsF comprises an nucleotide sequence having at least 70%, such as at least 75% sequence identity with SEQ ID NO: 94, and the gene epsG comprises an nucleotide sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 95.

According to some embodiments, the epsD gene comprises a nucleotide sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 92, the gene epsE comprises a nucleotide sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 93, the gene epsF comprises an nucleotide sequence having at least 80%, such as at least 85% sequence identity with SEQ ID NO: 94, and the gene epsG comprises an nucleotide sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 95.

According to some embodiments, the epsD gene comprises a nucleotide sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 92, the gene epsE comprises a nucleotide sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 93, the gene epsF comprises an nucleotide sequence having at least 90%, such as at least 95% sequence identity with SEQ ID NO: 94, and the gene epsG comprises an nucleotide sequence having at least 90%, such as at least 95% sequence identity with SEQ ID NO: 95.

According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 42, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 43, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 44, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 45.

According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 42, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 43, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 44, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 45.

According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 42, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 43, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 44, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 45.

According to some embodiments, the gene epsD comprises a nucleotide sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 128, the gene epsE comprises a nucleotide sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 129, the gene epsF comprises an nucleotide sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 130, and the gene epsG comprises an nucleotide sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 131.

According to some embodiments, the gene epsD comprises a nucleotide sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 128, the gene epsE comprises a nucleotide sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 129, the gene epsF comprises an nucleotide sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 130, and the gene epsG comprises an nucleotide sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 131.

According to some embodiments, the gene epsD comprises a nucleotide sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 128, the gene epsE comprises a nucleotide sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 129, the gene epsF comprises an nucleotide sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 130, and the gene epsG comprises an nucleotide sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 131.

According to some embodiments, the genetically engineered bacterium of the invention has been modified to inactivate the first EPS gene cluster.

According to some embodiments, the genetically engineered bacterium of the invention has been (further) modified to inactivate the second EPS gene cluster.

According to some embodiments, the genetically engineered bacterium of the invention has been modified to inactivate the first EPS gene cluster and second EPS gene cluster.

According to some embodiments, the first EPS gene cluster is inactivated by deletion of part of or the entire sequence of said cluster.

According to some embodiments, the first EPS gene cluster is inactivated by deletion of the entire coding sequence of said cluster.

According to some embodiments, the first EPS gene cluster is inactivated by deletion of the entire sequence of said cluster.

According to some embodiments, the first EPS gene cluster is inactivated by modification of (e.g., by introducing at least one mutation in) the promoter and/or ribosome binding site region resulting in the lack of gene expression.

According to some embodiments, the second EPS gene cluster is inactivated by deletion of part of or the entire sequence of said cluster.

According to some embodiments, the second EPS gene cluster is inactivated by deletion of the entire coding sequence of said cluster.

According to some embodiments, the second EPS gene cluster is inactivated by deletion of the entire sequence of said cluster.

According to some embodiments, the second EPS gene cluster is inactivated by modification of (e.g., by introducing at least one mutation in) the promoter and/or ribosome binding site region resulting in the lack of gene expression.

According to some embodiments, the genetically engineered bacterium of the invention has been modified to delete a nucleotide sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 181 or 183. According to some embodiments, the genetically engineered bacterium of the invention has been modified to delete a nucleotide sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 181 or 183. According to some embodiments, the genetically engineered bacterium of the invention has been modified to delete a nucleotide sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 181 or 183.

According to some embodiments, the genetically engineered bacterium of the invention has been modified to delete a nucleotide sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 182 or 184. According to some embodiments, the genetically engineered bacterium of the invention has been modified to delete a nucleotide sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 182 or 184. According to some embodiments, the genetically engineered bacterium of the invention has been modified to delete a nucleotide sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 182 or 184.

According to some embodiments, the genetically engineered bacterium of the invention has been modified to delete a nucleotide sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 181 and to delete a nucleotide sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 182. According to some embodiments, the genetically engineered bacterium of the invention has been modified to delete a nucleotide sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 181 and to delete a nucleotide sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 182. According to some embodiments, the genetically engineered bacterium of the invention has been modified to delete a nucleotide sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 181 and to delete a nucleotide sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 182.

According to some embodiments, the genetically engineered bacterium of the invention has been modified to delete a nucleotide sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 183 and to delete a nucleotide sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 184. According to some embodiments, the genetically engineered bacterium of the invention has been modified to delete a nucleotide sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 183 and to delete a nucleotide sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 184. According to some embodiments, the genetically engineered bacterium of the invention has been modified to delete a nucleotide sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 183 and to delete a nucleotide sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 184.

According to some embodiments, the genetically engineered bacterium of the invention has been modified to delete all endogenous genes encoding polypeptides involved in the production of exopolysaccharides (EPS) in said bacterium.

A bacterium in accordance with the present invention can be produced from any suitable bacterium of the genus Methylobacillus. Methylobacillus is a genus of Gram-negative methylotrophic bacteria.

According to some embodiments, the bacterium of the present invention is selected from Methylobacillus flagellatus, Methylobacillus glycogenes, Methylobacillus pratensis and Methylobacillus rhizosphaerae.

According to some embodiments, the bacterium of the present invention is selected from Methylobacillus flagellates and Methylobacillus glycogenes.

According to some embodiments, the bacterium of the present invention is Methylobacillus flagellatus.

According to some embodiments, the bacterium of the present invention is Methylobacillus glycogenes.

Method of the Invention

The present invention also provides methods for producing a biochemical compound comprising cultivating a bacterium according to the invention under suitable culture conditions in a culture medium comprising a reduced one-carbon compound, such as methanol, or a multi-carbon compound that contain no carbon-carbon bonds, such as dimethylamine.

The culture medium employed may be any conventional medium suitable for culturing a bacterium cell in question, and may be composed according to the principles of the prior art. The medium will usually contain all nutrients necessary for the growth and survival of the respective bacterium, such as carbon and nitrogen sources and other inorganic salts. Suitable media, e.g. minimal or complex media, are available from commercial suppliers, or may be prepared according to published receipts, e.g. the American Type Culture Collection (ATCC) Catalogue of strains. Non-limiting standard medium well known to the skilled person include Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, MS broth, Yeast Peptone Dextrose, BM MY, GM MY, or Yeast Malt Extract (YM) broth, which are all commercially available. A non-limiting example of suitable media for culturing bacterial cells, such as E. coli cells, including minimal media and rich media such as Luria Broth (LB), M9 media, M17 media, SA media, MOPS media, Terrific Broth, YT and others.

The carbon source may be any suitable carbon substrate known in the art, and in particularly any carbon substrate commonly used in the cultivation of methylotrophic bacteria and/or fermentation. A carbon source of particular interest is a reduced one-carbon compound, such as methanol or methylamine, or a multi-carbon compound that contains no carbon-carbon bonds, such as dimethylamine. Thus, according to some embodiments, the culture medium comprises methanol as a carbon source. The concentration of methanol in the culture medium may generally be in the range from about 0,5% w/v to about 4% w/v, such as from about 2% w/v to about 4% w/v. According to some embodiments, the concentration of methanol in the culture medium is in the range from about 2,5% w/v to about 3,5% w/v.

As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate, and digested fermentative microorganism can be used. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used.

The cultivation can be preferably performed under aerobic conditions, such as by a shaking culture, by a stirring culture or in a bioreactor with aeration, at a temperature of about 20 to about 45° C., such as about 30 to 38° C., preferably at about 37° C. The pH of the culture is usually above 5, such as in a range from about 6 to about 8, preferably from about 6.5 to about 7.5, more preferably from about 6.8 to about 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. The cultivation may be carried out for a period in the range from 10 to 70 h, preferably in a range from 24 to 60 h, more preferably in a range from 36 to 50 h.

After cultivation, solids such as cells can be removed from the culture medium by centrifugation or membrane filtration. The biochemical compound can be collected by conventional method for isolation and purification chemical compounds from a medium. Well-known purification procedures include, but are not limited to, centrifugation or filtration, precipitation, ion exchange, chromatographic methods such as e.g. ion exchange chromatography or gel filtration chromatography, and crystallization methods. The method may further comprise collecting the biochemical compound from the culture medium.

The biochemical compound produced by the method of the present invention may be any desired compound obtained through the cultivation of a bacterium of the present invention. Non-limiting examples include organic acids, amino acids, fatty acids and derivatives thereof. Non-limiting examples of organic acids include fumarate, glycolate, succinate, malate, malonate, lactates and derivatives thereof. Non-limiting examples of amino acids include glutamate, lysine, methionine, tryptophan, phenylalanine and derivatives thereof.

The present invention thus provides a biochemical compound obtainable by a method as detailed herein.

Certain Other Definitions

“Polypeptide” and “protein” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids.

“Nucleic acid” or “polynucleotide” are used interchangeably herein to denote a polymer of at least two nucleic acid monomer units or bases (e.g., adenine, cytosine, guanine, thymine) covalently linked by a phosphodiester bond, regardless of length or base modification.

“Recombinant” or “non-naturally occurring” when used with reference to, e.g., a host cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant bacterial cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.

“Heterologous” or “exogenous” as used herein in the context of a gene or nucleic acid molecule refer to a gene or nucleic acid molecule (i.e. DNA or RNA molecule) that does not occur naturally as part of the genome of the bacterium in which it is present or which is found in a location or locations in the genome that differ from that in which it occurs in nature. Thus, a “heterologous” or “exogenous” gene or nucleic acid molecule is not endogenous to the bacterium and has been exogenously introduced into the microorganism. A “heterologous” gene or nucleic acid molecule DNA molecule may be from a different organism, a different species, a different genus or a different kingdom, as the host DNA.

“Heterologous” as used herein in the context of a polypeptide means that a polypeptide is normally not found in or made (i.e. expressed) by the host microorganism, but derived from a different organism, a different species, a different genus or a different kingdom.

As used herein, the term “ortholog” or “orthologs” refers to genes, nucleic acid molecules encoded thereby, i.e., mRNA, or proteins encoded thereby that are derived from a common ancestor gene but are present in different species.

By “decreased expression” of a gene it is meant that the amount of the transcription product, respectively the amount of the polypeptide (e.g., enzyme) encoded by said gene produced by the modified bacterium is decreased compared to an otherwise identical bacterium that does not carry said modification. More particularly, by “decreased expression” of a gene it is meant that the amount of the transcription product, respectively the amount of the polypeptide (e.g., enzyme) encoded by said gene produced by the modified bacterium is decreased by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50% at least 60%, at least 70%, at least 80%, at least 90% or at least 100%, compared to an otherwise identical bacterium that does not carry said modification. The level of expression of a gene can be determined by well-known methods, including PCR, Southern blotting, and the like. In addition, the level of gene expression can be estimated by measuring the amount of mRNA transcribed from the gene using various well-known methods, including Northern blotting, quantitative RT-PCR, and the like. The amount of the polypeptide encoded by the gene can be measured by well-known methods, including ELISA, Immunohistochemistry or Western Blotting and the like.

Expression of a gene can be decreased by introducing a mutation into the gene in the genome of the bacterium so that the intracellular activity of the polypeptide encoded by the gene is decreased as compared to an otherwise identical bacterium that does not carry said mutation. Mutations which result in a decreased expression of the gene include the replacement of one nucleotide or more to cause an amino acid substitution in the polypeptide encoded by the gene (missense mutation), introduction of a stop codon (nonsense mutation), deletion or insertion of nucleotides to cause a frame shift, insertion of a drug-resistance gene, or deletion of a part of the gene or the entire gene (Qiu and Goodman, 1997; Kwon et al., 2000). Expression can also be decreased by modifying an expression regulating sequence such as the promoter, the Shine-Dalgarno (SD) sequence, etc. Expression of the gene can also be decreased by gene replacement (Datsenko and Wanner, 2000), such as the “lambda-red mediated gene replacement”. The lambda-red mediated gene replacement is a particularly suitable method to inactive one or more genes as described herein.

“Inactivating”, “inactivation” and “inactivated”, when used in the context of a gene or gene cluster, means that the gene or gene cluster in question no longer expresses a functional protein. It is possible that the modified DNA region is unable to naturally express the gene or gene cluster due to the deletion of a part of or the entire sequence of the gene or gene cluster, the shifting of the reading frame of the gene or gene cluster, the introduction of missense/nonsense mutation(s), or the modification of the regulatory region of the gene or gene cluster, including sequences controlling gene expression, such as a promoter, enhancer, attenuator, ribosome-binding site, etc. Preferably, a gene or gene cluster of interest is inactivated by deletion of a part of or the entire sequence of the gene or gene cluster, such as by gene replacement. Inactivation may also be accomplished by introducing or expressing a rare-cutting endonuclease able to selectively inactivating by DNA cleavage, preferably by double-strand break, the gene or gene cluster of interest. A “rare-cutting endonuclease” within the context of the present invention includes transcription activator-like effector (TALE) nucleases, meganucleases, zing-finger nucleases (ZFN), and RNA-guided endonucleases.

The presence or absence of a gene or gene cluster in the genome of a bacterium can be detected by well-known methods, including PCR, Southern blotting, and the like. In addition, the level of gene expression can be estimated by measuring the amount of mRNA transcribed from the gene or gene cluster using various well-known methods, including Northern blotting, quantitative RT-PCR, and the like. The amount of the polypeptide encoded by the gene or gene cluster can be measured by well-known methods, including SDS-PAGE followed by an immunoblotting assay (Western blotting analysis), and the like.

As used herein, “decreased”, “decreasing” or “decrease of” expression of a polypeptide (such as an enzyme as described herein) means that the expression of said polypeptide in a modified bacterium is reduced compared to the expression of said polypeptide in an otherwise identical bacterium that does not carry said modification (control). The expression of a polypeptide in a modified bacterium may be reduced by at least about 10%, and preferably by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100%, or any percentage, in whole integers between 10% and 100% (e.g., 6%, 7%, 8%, etc.), compared to the expression of said polypeptide in an otherwise identical bacterium that does not carry said modification (control). More particularly, “decreased”, “decreasing” or “decrease of” expression of a polypeptide means that the amount of the polypeptide in the modified bacterium is reduced by at least about 10%, and preferably by at least about 20%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100%, or any percentage, in whole integers between 10% and 100% (e.g., 6%, 7%, 8%, etc.), compared to the amount of said polypeptide in an otherwise identical bacterium that does not carry said modification (control). The expression or amount of a polypeptide in a bacterium can be determined by any suitable means know in the art, including techniques such as ELISA, Immunohistochemistry, Western Blotting or Flow Cytometry.

As used herein, “abolished” expression of a polypeptide (such as an enzyme as described herein) means that the expression of said polypeptide in a modified bacterium is not detectable compared to the expression of said polypeptide in an otherwise identical bacterium that does not carry said modification (control).

As used herein, “decreased”, “decreasing” or “decrease of” activity of a polypeptide (such as an enzyme as described herein) means that the catalytic activity of said polypeptide in a modified bacterium is reduced compared to the catalytic activity of said polypeptide in an otherwise identical bacterium that does not carry said modification (control). The activity of a polypeptide in a modified bacterium may be reduced by at least about 10%, and preferably by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100%, or any percentage, in whole integers between 10% and 100% (e.g., 6%, 7%, 8%, etc.), compared to the expression of said polypeptide in an otherwise identical bacterium that does not carry said modification (control). The activity of a polypeptide in a bacterium can be determined by any suitable protein and enzyme activity assay.

“Expression” includes any step involved in the production of a polypeptide (e.g., encoded enzyme) including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

As used herein, “regulatory region” of a gene or gene cluster refers to a nucleic acid sequence that affect the expression of a coding sequence. Regulatory regions are known in the art and include, but are not limited to, promoters, enhancers, transcription terminators, polyadenylation sites, matrix attachment regions and/or other elements that regulate expression of a coding sequence.

“Substitution” or “substituted” refers to modification of the polypeptide by replacing one amino acid residue with another, for instance the replacement of an Serine residue with a Glycine or Alanine residue in a polypeptide sequence is an amino acid substitution. When used with reference to a polynucleotide, “substitution” or “substituted” refers to modification of the polynucleotide by replacing one nucleotide with another, for instance the replacement of a cytosine with a thymine in a polynucleotide sequence is a nucleotide substitution.

“Conservative substitution”, when used with reference to a polypeptide, refers to a substitution of an amino acid residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid, e.g., alanine, valine, leucine, and isoleucine; an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain, e.g., serine and threonine; an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with a basic side chain is substituted with another amino acid with a basic side chain, e.g., lysine and arginine; an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain, e.g., aspartic acid or glutamic acid; and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

“Non-conservative substitution”, when used with reference to a polypeptide, refers to a substitution of an amino acid in a polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., serine for glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

As used herein, “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded nucleic acid loop into which additional nucleic acid segments can be ligated. Certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. Certain other vectors are capable of facilitating the insertion of an exogenous nucleic acid molecule into a genome of a bacterium. Such vectors are referred to herein as “transformation vectors”. In general, vectors of utility in recombinant nucleic acid techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of a vector. Large numbers of suitable vectors are known to those of skill in the art and commercially available.

As used herein, “promoter” refers to a sequence of DNA, usually upstream (5′) of the coding region of a structural gene, which controls the expression of the coding region by providing recognition and binding sites for RNA polymerase and other factors which may be required for initiation of transcription. The selection of the promoter will depend upon the nucleic acid sequence of interest. A suitable “promoter” is generally one which is capable of supporting the initiation of transcription in a bacterium of the invention, causing the production of an mRNA molecule.

As used herein, “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence. A promoter sequence is “operably-linked” to a gene when it is in sufficient proximity to the transcription start site of a gene to regulate transcription of the gene.

“Percentage of sequence identity,” “% sequence identity” and “percent identity” refers to sequence identity between a nucleotide sequence and a reference nucleotide sequence or between an amino acid sequence and a reference amino acid sequence. Sequence identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of identity between nucleotide or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the nucleotide or amino acid sequences, respectively. Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with default settings.

As used herein, the term “about” means plus or minus 10% of the numerical value of the number with which it is being used.

Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and sub ranges within a numerical limit or range are specifically included as if explicitly written out.

As used herein, the indefinite articles “a” and “an” mean “at least one” or “one or more” unless the context clearly dictates otherwise.

As used herein, the terms “comprising”, “including”, “having” and grammatical variants thereof are to be taken as specifying the stated features, steps or components but do not preclude the addition of one or more additional features, steps, components or groups thereof.

Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

EXAMPLES Example 1: Identification of EPS-Producing Genes in Methylobacillus flagellatus and Methylobacillus glycogenes

Several genomic loci were chosen for disruption based on bioinformatic analysis. The genetic locations of two EPS-associated gene clusters in Methylobacillus flagellatus have been published previously (Chistoserdova et al., 2007). To annotate genes in the first Methylobacillus flagellatus EPS-producing gene cluster (SEQ ID NOs: 87-112), homologues for genes from the published Methylobacillus sp. 12S methanolan synthesis cluster (Yoshida et al., 2000) were compared to the Methylobacillus flagellatus genome via an NCBI BLAST protein search, using a custom written script in the Python programming language. Top hits were selected based on a computed combination-score consisting of E-value, query coverage and sequence identity. A single top hit for each gene was then manually located in the genome sequence and annotated. Locations of these top hits coincided with the location of the published first cluster. Since a clear homologue was not found for each gene, all genes between the most distant homologues were considered to be part of the cluster. Most such non-annotated genes coded for sugar and polysaccharide related genes. The same procedure was used to identify the completely non-annotated EPS cluster in Methylobacillus glycogenes (SEQ ID NOs: 125-146).

A second EPS-related cluster was predicted in the genome sequence of Methylobacillus flagellatus (SEQ ID NOs: 113-124). A BLAST search comparing these genes to the methanolan cluster was performed, finding no clear homologues. The protein product of each individual gene in this cluster was identified via a NCBI protein BLAST search against all non-redundant protein sequences and the top hit for each was chosen as described above. The protein sequence of each gene from this Methylobacillus flagellatus protein cluster was cross-searched in the Methylobacillus glycogenes genome using NCBI protein BLAST. The top hits were found to form a similar cluster in Methylobacillus glycogenes (SEQ ID NOs: 147-172).

The identified open reading frames (ORFS) in each EPS-related gene cluster are shown in Tables 1 to 4 below.

TABLE 1 ORFs of EPS gene cluster 1 in M. flagellatus Amino acid Nucleotide sequence sequence S12 SEQ ID NO SEQ ID NO homolog Predicted function/property 1 87 espA Transcriptional regulator, LuxR family 2 88 espK Cyclic nucleotide-binding domain (cNMP-BD)protein 3 89 N/A Unknown 4 90 epsL Exosortase B-associated extracellular polysaccharide biosynthesis transporter EpsL 5 91 epsB Transferase enzyme epsB family 6 92 epsD Peptidyl-prolyl cis-trans isomerase, EpsD family 7 93 epsE Polysaccharide export protein EpsE 8 94 epsF Chain length determinant protein EpsF 9 95 epsG Chain length determinant protein tyrosine kinase EpsG 10 96 epsH Exosortase B 11 97 epsI EpsI family protein 12 98 N/A Polysaccharide biosynthesis protein 13 99 N/A Polysaccharide pyruvyl transferase 14 100 N/A Nitroreductase 15 101 N/A O-antigen polymerase 16 102 epsJ Glycosyl transferase, family 2 17 103 N/A Glycosyl transferase, family 2 18 104 N/A Glycosyl transferase, group 1 19 105 N/A Glycoside hydrolase, family 5 20 106 N/A Glycosyltransferase, RfaG 21 107 N/A Mannose-1-phosphate guanylyltransferase (GDP) 22 108 N/A dTDP-4-dehydrorhamnose reductase 23 109 N/A dTDP-4-dehydrorhamnose 3,5-epimerase 24 110 N/A Glucose-1-phosphate thymidylyltransferase 25 111 N/A dTDP-glucose 4,6-dehydratase 26 112 epsS UDP-galactose 4-epimerase

TABLE 2 ORFs of EPS gene cluster 2 in M. flagellatus Amino acid Nucleotide sequence sequence S12 SEQ ID NO SEQ ID NO homolog Predicted function/property 27 113 N/A Mannose-6-phosphate isomerase, type 2 28 114 N/A ABC transporter permease 29 115 N/A NDP-hexose 3-C-methyltransferase TylCIII 30 116 N/A GDP-mannose 4,6-dehydratase 31 117 N/A NAD-dependent epimerase/dehydratase 32 118 N/A Glycosyl transferase 33 119 N/A Polysaccharide export protein 34 120 N/A Glycosyl transferase 35 121 N/A NAD-dependent epimerase/dehydratase 36 122 N/A Sugar transferase 37 123 N/A Polysaccharide biosynthesis protein CapD 38 124 N/A UDP-glucose pyrophosphorylase

TABLE 3 ORFs of EPS gene cluster 2 in M. glycogenes Amino acid Nucleotide sequence sequence S12 SEQ ID NO SEQ ID NO homolog Predicted function/property 39 125 epsA Transcriptional regulator, LuxR family 40 126 epsL Exosortase B-associated extracellular polysaccharide biosynthesis transporter EpsL 41 127 N/A Undecaprenyl-phosphate glucosephosphotransferase 42 128 epsD Peptidyl-prolyl cis-trans isomerase, EpsD family 43 129 epsE Polysaccharide export protein EpsE 44 130 epsF Chain length determinant protein EpsF 45 131 epsG Chain length determinant protein tyrosine kinase EpsG 46 132 epsH Exosortase B 47 133 epsI EpsI family protein 48 134 epsB Glycosyltransferase 49 135 N/A Oligosaccharide repeat unit polymerase 50 136 N/A Right-handed parallel beta-helix repeat-containing protein 51 137 N/A Acyltransferase family protein 52 138 N/A Glycosyltransferase family 4 protein 53 139 N/A Glycosyltransferase family 4 protein 54 140 N/A Nucleotide sugar dehydrogenase 55 141 epsR Unknown 56 142 N/A Unknown 57 143 N/A Oligosaccharide flippase family protein 58 144 N/A Polysaccharide pyruvyl transferase family protein 59 145 N/A Acyltransferase 60 146 epsQ mannose-1-phosphate guanylyltransferase/mannose- 6-phosphate isomerase

TABLE 4 ORFs of EPS gene cluster 2 in M. glycogenes Amino acid Nucleotide sequence sequence S12 SEQ ID NO SEQ ID NO homolog Predicted function/property 61 147 N/A MarR family EPS-associated transcriptional regulator 62 148 N/A GDP-mannose 4,6-dehydratase 63 149 N/A NAD-dependent epimerase/dehydratase family protein 64 150 N/A Unknown 65 151 N/A Polyhydroxyalkanoate synthesis repressor PhaR 66 152 N/A UDP-N-acetylglucosamine 2-epimerase (hydrolyzing) neuC 67 153 N/A Imidazole glycerol phosphate synthase subunit HisF 68 154 N/A Imidazole glycerol phosphate synthase subunit HisH 69 155 N/A N-acetyl sugar amidotransferase 70 156 N/A Polysaccharide pyruvyl transferase family protein 71 157 N/A Acyltransferase 72 158 N/A Unknown 73 159 N/A ABC transporter ATP-binding protein 74 160 N/A FkbM family methyltransferase 75 161 N/A Glycosyltransferase 76 162 N/A Methyltransferase, TIGR04325 family 77 163 N/A NAD-dependent epimerase/dehydratase family protein 78 164 N/A DegT/DnrJ/EryC1/StrS family aminotransferase 79 165 N/A Glycosyltransferase 80 166 epsJ Glycosyltransferase 81 167 N/A Glycosyltransferase 82 168 N/A Glycosyltransferase 83 169 N/A SDR family oxidoreductase 84 170 N/A Sugar transferase 85 171 N/A SDR family NAD(P)-dependent oxidoreductase 86 172 epsT UTP--glucose-1-phosphate uridylyltransferase GalU

TABLE 5 Full sequences of clusters SEQ ID NO Organism Cluster Strand 173 M. flagellatus 1 top 174 M. flagellatus 1 bottom 175 M. flagellatus 2 top 176 M. flagellatus 2 bottom 177 M. glycogenes 1 top 178 M. glycogenes 1 bottom 179 M. glycogenes 2 top 180 M. glycogenes 2 bottom

ORFS annotated as epsD, epsE, epsF and epsG (SEQ ID NO: 6-9 in M. flagellatus, SEQ ID NO: 42-45 in M. glycogenes) were chosen for deletion, based on their similarity to EPS-production essential genes in Methylobacillus sp. strain 12 (Yoshida et al., 2003). Since the second EPS cluster had no clear homologues in other characterized EPS clusters, all 12 ORFS in M. flagellatus (SEQ ID NO: 27-38) and all 26 ORFS in Methylobacillus glycogenes (SEQ ID NO: 61-86) were chosen for deletion.

Example 2: DNA Deletions

Modified strains of ABME 5 and ABME 6 were preparing in which either one EPS-producing cluster or both EPS-producing clusters are deleted. The genome edits were achieved by introducing a linear DNA fragment consisting a kanamycin or rifampicin resistance cassette flanked by 2 kb long sequences upstream and downstream of the integration sites. DNA fragments were transformed into ABME 5 and ABMR 6 by electroporation. Transformations were plated onto selective PM7 plates containing 20 g/L agar, 50 μg/mL kanamycin, or 5 μg/mL rifampicin for 24-48 h. ABME 5-derived strains were incubated at 30° C. ABME 6-derived strains were cultivated at 37° C. Correct clones were confirmed by colony PCR. The strains prepared by DNA deletion are listed in Table 6.

TABLE 6 List of strains prepared with genomic integration. Strain Species Genotype Deleted sequences ABME Methylobacillus Wild type 5 glycogenes DSM 5685 ABME Methylobacillus epsDEFG :: kanR SEQ ID NO: 181 76 glycogenes DSM 5685 ABME Methylobacillus EPS2 :: kanR SEQ ID NO: 182 190TH glycogenes DSM 5685 ABME Methylobacillus epsDEFG :: kanR, SEQ ID NO: 181 and 191TH glycogenes EPS2 :: rifR SEQ ID NO: 182 DSM 5685 ABME Methylobacillus Wild type 6 flagellatus DSM 6875 ABME Methylobacillus EPS2 :: kanR SEQ ID NO: 184 80 flagellatus DSM 6875 ABME Methylobacillus epsDEFG :: kanR SEQ ID NO: 183 82 flagellatus DSM 6875 ABME Methylobacillus epsDEFG :: kanR, SEQ ID NO: 183 and 131 flagellatus EPS2 :: rifR SEQ ID NO: 184 DSM 6875 ABME Methylobacillus Wild type 145 sp. DSM 1758

Example 3: Measurements of Total EPS in Shake Flask Cultures before and after EPS Gene Disruption in Methylobacillus flagellatus

To evaluate the effects of deleting EPS-producing genes in ABME 6, three modified ABME 6 strains (ABME 80, ABME 82 and ABME 131) were prepared as described in Example 3 and tested for EPS production.

Confirmed transformants were grown overnight on PM7 +50 μg/mL kanamycin agar plates. Single colonies were resuspended in 25 mL PM7 medium+25 μg/mL kanamycin in baffled 250 mL shake flasks, and incubated overnight at 200 rpm. Next day, 50 mL PM7 medium+25 μg/mL kanamycin in baffled 250 mL shake flasks was inoculated with 2.5 mL overnight culture. All ABME 5-derived strains were cultivated at 30° C., and all ABME 6-derived strains were cultivated at 37° C. The cultures were then chilled on ice, aliquoted into 2 mL Eppendorf tubes and centrifuged for 15 minutes at 15 000 rpm on a standard tabletop centrifuge at 4° C. When a layer of EPS was visible above the cell pellet, it was mixed back into the supernatant. The culture supernatants were stored for EPS hydrolysis. Hydrolysis was performed by adding half the volume of the supernatant of 6M TFA (trifluoroacetic acid), resulting in a final TFA concentration of 3M. Tubes were placed into a thermoblock heated to 120° C. and incubated for 6h. After hydrolysis, the liquid (including TFA) was evaporated at 80° C. until completely dry. The dry mass was resuspended in 0.5 M NaOH (at the same volume as the original culture aliquot) and analyzed for monomeric sugars on an HPLC.

TABLE 7 Composition of PM7 medium used for shake-flask experiments Compound Per 1 L MeOH 10 g KH2PO4 0.65 g NA2HPO4 1.05 g MgSO4 * 7H2O 0.1 g NH4Cl 0.2 g (NH4)2SO4 2 g TE1 1000X 1 mL d-Biotin 0.1 mg Thiamine*HCl 0.1 mg Riboflavin 0.1 mg Pyridoxine*HCl 0.1 mg Pantothenate 0.1 mg Nicotinamide 0.1 mg p-Aminobenzoic acid 0.02 mg Folic acid 0.01 mg Vitamin B12 0.01 mg Lipoic acid 0.01 mg CaCl2 * 2H2O 3.3 mg FeSO4 * 7H2O 1.3 mg MnSO4 * H2O 0.1 mg ZnSO4 * 7H2O 0.13 mg CuSO4 * 5H2O 0.04 mg Na2MoO4 * 2H2O 0.04 mg CoCl2 * 6H2O 0.04 mg H3BO3 0.03 mg pH 7.0 Agar (for solid media) 20 g

ABME 6 produced a visible slime layer on top of the cell pellet after centrifugation, amounting to ˜1 g/L total monomeric sugars after hydrolysis. The single-cluster disruptions (ABME 80 and ABME 82) had no visible slime layer, but still produced ˜500 mg/L total sugars. Interestingly, the glucose to total sugars ratio is inverted depending on which gene cluster was disrupted, most likely due to two different types of EPS being produced. When both clusters were disrupted (strain ABME 131), sugars were below the detection limit.

Example 4: Measurements of Viscosity in Reactor Cultures of Methylobacillus flagellatus before and after EPS Gene Disruption

To assess the change in fermentation broth viscosity before and after the disruption of EPS production, the strains were compared in 5-liter bioreactor fermentations under oxygen limitation. The bioreactor seed cultures were always prepared as follows. 25 mL of PM7 medium in 250 mL baffled shake-flasks were inoculated with 250 uL of strain cryo-stock with the stock OD adjusted to 5, to produce a starting OD of 0.05. The cultures were incubated overnight at 200 rpm 37° C. In the morning, 200 mL of PM7 medium in 2 L baffled shake-flasks was inoculated with 10% (20 mL) of the overnight culture and grown until OD ˜1 (+/−0.2). The culture was then used to inoculate a 2.5 L starting volume of sterile PM3 medium in 5-L bioreactors (8% inoculum). The pH was kept at 7 by automatic addition of NH₄OH and the temperature was kept at 37° C.

For oxygen limited processes, methanol was fed automatically to maintain a concentration between 3 and 6 g/L. Reactor aeration and stirring were kept at maximum, though the oxygen concentration always dropped to 0% during the process. The broth was sampled regularly for OD, mineral and metabolite measurements. Viscosity was measured at the end of the process and the results are summarized in FIG. 2 . Wild type M. flagellatus fermentation broth was highly viscous at the time of sampling at 400 centipoise. A different species of Methylobacillus, ABME 145 was measured as well and produced a similarly viscous broth of 422 centipoise. The double disruption produced a visibly less viscous broth, which could be centrifuged and filtered using standard tabletop equipment—something that was not possible with the previous samples. The viscosimeter confirmed this assessment, measuring in at 35 cP or a nearly 10-fold reduction in viscosity. A representative sample of Escherichia coli fermentation broth was also measured for comparison.

Example 5: Measurements of Reduced Foaming in Shake Flasks before and after EPS Gene Elimination in Methylobacillus flagellates

We observed that the EPS-deficient strains showed significantly less foaming. To compare foam formation of ABME 6 against EPS-deficient mutant strains (ABME 82 and ABME 131), the strains were culture as described in Example 3.

To measure foam formation flasks were taken off the shaker and the liquid was allowed to settle for 5 minutes. The foam layer on top of the liquid was measured with a tape measure (FIG. 3 ). Wild type cultures produced 0.5-1 cm of foam layer on top of the liquid while the single-cluster disruptions only accumulated ˜1 mm of foam at the liquid meniscus. No foam at all was visible in the double-disruption cultures.

Example 6: Measurements of Reduced Foaming in Bioreactor Cultivation before and after EPS Gene Disruption in Methylobacillus flagellatus

To assess the change in fermentation broth foaming before and after the disruption of EPS production, the strains were compared in 5-liter bioreactor fermentations. The amount of foam formation was approximated by the amount of added anti-foaming agent (SAG 5693). The fermentations were carried out under carbon limitation.

The reactors were inoculated as described in Example 5. To achieve carbon limitation, 50% methanol was fed to the reactor automatically at a dynamically-calculated rate that that maintained pO2-value at approximately 30% at the highest aeration and stirring settings. The added methanol was consumed immediately and kept below detection levels. Antifoam was added automatically when foam reached a detector placed above the fermentation broth meniscus. ABME 6 and ABME 131 were tested in this way. Antifoam addition started 5 h later in the case of ABME 131. Approximately 60% less antifoam was added in the ABME 131 fermentation, depending on the fermentation time (FIG. 4 ).

Example 7: Microscope Images of Methylobacillus flagellatus Cell Aggregates before and after the EPS Gene Disruption

To assess the change in cell clumping foaming before and after the disruption of EPS production, the strains were cultured in 5-liter bioreactor fermentations under oxygen limitation as described in Example 3.

Samples of broth were observed by phase-constrast microscopy on a Zeiss Axioplan 2 microscope through a Zeiss Ph2 Plan-NEOFLUAR 40× objective (NA 0.75) and recorded on a sCMOS camera. Cells of ABME 6 formed large clumps, and were immobilized in the EPS matrix (FIG. 5 ). In the case of the double-disruption strain ABME 131, cells floated individually in solution, with minimal clumping.

Example 8: Improved Methanol Tolerance of EPS-Free M. flagellates

Tolerance to high methanol concentrations is a critical parameter for efficient bioprocesses. To confirm that methanol tolerance was not reduced in strains that cannot produced EPS, growth at increasing methanol concentrations was tested in ABME 6 and its EPS-producing derivative strain ABME 131.

The strains were tested in shake flasks as described in Example 4. To test methanol tolerance, the initial methanol concentration was increased up to 4.5%. OD600 was measured after 24 h.

Surprisingly, the methanol tolerance of ABME 131 was higher compared to ABME 6. For example, at 2.75% initial methanol concentration, the EPS-free ABME 131 grew to an OD600 of 1.6, whereas the wild-type ABME 6 only grew to an OD600 of 0.3. The difference in growth was most striking at 2.75%. However improved growth of ABME 131 compared to ABME 6 was apparent at all methanol concentration (FIG. 6 ).

LIST OF REFERENCES CITED IN THE DESCRIPTION

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1. A genetically engineered bacterium of the genus Methylobacillus which has been modified to inactivate an EPS gene cluster which includes genes defined by or orthologous to the open reading frames (ORFs) found in SEQ ID NO: 175 or
 179. 2-22. (canceled)
 23. The bacterium according to claim 1, wherein an additional EPS gene cluster is inactivated and, wherein said additional EPS gene cluster includes genes defined by or orthologous to the open reading frames (ORFs) found in SEQ ID NO: 174 or
 177. 24. The bacterium according to claim 23, wherein at least one gene in said additional EPS gene cluster is inactivated.
 25. The bacterium according to claim 23, wherein said additional EPS gene cluster includes the genes epsD, epsE, epsF and epsG, or orthologs thereof.
 26. The bacterium according to claim 25, wherein the genes epsD, epsE, epsF and epsG, or orthologs thereof, are inactivated.
 27. The bacterium according to claim 25, wherein the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6, wherein the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 7, wherein the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8, and wherein the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 9; or wherein the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 42, wherein the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 43, wherein the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 44, and wherein the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:
 45. 28. The bacterium according to claim 1, wherein an additional EPS gene cluster is inactivated.
 29. The bacterium according to claim 1, wherein said EPS gene cluster is inactivated.
 30. The bacterium according to claim 28, wherein said additional EPS gene cluster and/or said EPS gene cluster is inactivated by deletion of part of or the entire sequence of said cluster or by modification of the promoter and/or ribosome binding site region resulting in the lack of gene expression.
 31. The bacterium according to claim 1, wherein the bacterium is Methylobacillus flagellatus.
 32. The bacterium according to claim 1, wherein the bacterium is Methylobacillus glycogenes.
 33. A method for the production of a biochemical compound comprising cultivating a bacterium according claim 1 under suitable culture conditions in a culture medium comprising a reduced one-carbon compound or a multi-carbon compound that contains no carbon-carbon bonds.
 34. The method according to claim 33, wherein said reduced one-carbon compound is methanol.
 35. The method according to claim 33, wherein said multi-carbon compound is dimethylamine. 